Specific, active immunotherapy is an attractive approach for the treatment of cancer. It is less toxic than chemotherapy and elicits immune surveillance mechanisms with the potential of providing protection from tumor recurrence. The long-term objective of this study is to develop an effective immunotherapy, targeting multiple epitopes of the tumor associated antigen, Her-2/neu. Our strategy employs a multi-allelic approach (HLA-A1, A2, and Cw7), expanding the patient population it will benefit. Targeting multiple alleles, including an HLA-C allele, significantly decreases the chances of immune escape by the tumor. Specifically, we aim to design, discover and generate a panel of modified Her-2/neu peptides containing both L and D-amino acids, capable of stimulating anti-Her-2/neu cytotoxic T lymphocytes (CTL) more strongly than wild type Her-2/neu peptide epitopes for HLA-A1, A2 and Cw7. Peptides will be rationally designed based upon our previous findings of preferred, residues at certain postitions. Using our novel, functional screening assay, we will also screen constrained, "residue-optimized" combinatorial peptide libraries containing D- and L-amino acids for peptides that induce cytokine (IFN-g) release from anti-Her-2/neu CTL. These peptides will be evaluated for the ability to stimulate stronger interferon-gamma secretion from CTL generated against wild type peptides, than the wild type peptide itself. Binding affinities of the peptides for their respective HLA molecules will be determined and correlated with their biological activities (induction of IFN-gamma) secretion. Candidate peptides will be tested for the ability to generate CTL specific for modified Her-2/neu peptides, but also able to elicit cross-reactive CTL, capable of killing tumors expressing wild type Her-2/neu. In future studies, we intend to expand this multi-allelic approach to include other tumor associated antigens.